OXYTOCIN NEUROPHYSIN-I MESSENGER-RIBONUCLEIC-ACID IN BOVINE GRANULOSA-CELLS INCREASES AFTER THE LUTEINIZING-HORMONE (LH) SURGE AND IS STIMULATED BY LH INVITRO

Bovine granulosa cells express the oxytocin/neurophysin-I (OT/NP-I) gene and secrete OT in vitro. We have shown previously that bovine granulosa cells isolated from the preovulatory follicle after the LH surge secrete 20 times more OT over 5 days in culture than granulosa cells obtained before the surge. LH or FSH stimulates OT secretion in vitro by granulosa cells isolated before the LH surge. We also observed that granulosa cells of preovulatory follicles isolated before the LH surge respond to OT with an increase in progesterone secretion, suggesting that OT may be involved in regulating the follicular/luteal phase shift, or ovulation, in an autocrine fashion. The objective of this study was to determine whether the increase in OT secretion from granulosa cells after the LH surge is regulated at the level of mRNA accumulation, peptide synthesis, and/or peptide secretion. Bovine preovulatory follicles were obtained during the early follicular phase (approximately 36 h before the LH surge), during the midfollicular phase (approximately 12 h before the LH surge), or during the late follicular phase (after the LH surge). Total RNA isolated from granulosa cells and theca interna at the time of cell isolation or after culture with or without LH was subjected to Northern analysis for OT/NP-I mRNA and quantified by densitometry. OT/NP-I mRNA was not detectable or was barely detectable in granulosa cells collected during the early or midfollicular phase (n = 6 and n = 4 follicles, respectively), but a strong hybridization signal was obtained from RNA isolated after the LH surge (n = 5 follicles; P < 0.01). In contrast, OT/NP-I mRNA was not detectable in theca interna before or after the LH surge. Although OT/NP-I mRNA was not detectable in granulosa cells isolated 24 h after prostaglandin F2alpha injection, after 24 h in culture, a weak OT/NP-I mRNA hybridization signal was observed in RNA from granulosa cells in LH-containing cultures. After 72 h in culture, granulosa cells cultured in control, as well as in LH-containing medium, exhibited a strong signal for OT/NP-I mRNA, but granulosa cells treated with LH exhibited a stronger OT/NP-I hybridization signal than control cultures (P < 0.01). Theca interna did not yield any OT/NP-I hybridization signal initially, and none was induced in culture. Intracellular OT was low in granulosa cells isolated 24 and 48 h after prostaglandin F2alpha injection (34 +/- 13 and 29 +/- 7 pg/10(7) cells) and 64 times higher 20 h after the onset of estrus (2034 +/- 824 pg/10(7) cells; P < 0.05). During the early and midfollicular phase, OT in follicular fluid was low (78 +/- 21 and 61 +/- 16 pg/ml, respectively), whereas follicular fluid obtained after the LH surge had 6 times more OT (423 +/- 96 pg/ml; P < 0.01). In conclusion, OT/NP-I mRNA accumulation, OT synthesis, and OT secretion are tightly correlated in granulosa cells in vivo. The results suggest that the preovulatory gonadotropin surge stimulates the accumulation of OT/NP-I mRNA in bovine granulosa cells, leading to dramatically increased secretion of OT.